Structural characterisation of human proteinosis surfactant protein A

https://doi.org/10.1016/S0167-4838(00)00184-9Get rights and content

Abstract

Human surfactant protein-A (SP-A) has been purified from a proteinosis patient and characterised by a combination of automated Edman degradation and mass spectrometry. The complete protein sequence was characterised. The major part of SP-A was shown to consist of SP-A2 gene product, and only a small amount of SP-A1 gene product was shown to be present. A cysteine extension to the N-terminal was indicated by sequence data, but was not definitely proven. All proline residues in the Y position of Gly-X-Y in the collagen-like region were at least partially modified to hydroxy-proline, but no lysine residues were found to be modified. A complex N-linked glycosylation was found on Asn-187 showing great heterogeneity as variants from a mono-antennary to penta-antennary glycosylation with varying amounts of attached pentose were identified. The disulfide bridges in the carbohydrate recognition domain were identified to be in the 1–4, 2–3 pattern common for collectins. Interchain disulfide bridges were discovered between two Cys-48 residues and cysteine residues in the N-terminal region. However, the exact disulfide bridge connections within the bouquet-like ultrastructure could not be established.

Introduction

Lung surfactant protein A (SP-A) belongs to the collectin group of proteins which are composed of C-type carbohydrate recognition domains (CRD) attached to collagen-like domains via α-helical neck regions. The individual chains are organised in trimeric subunits, which in most cases are combined as oligomers through N-terminally located cysteines. Six collectins have been described: SP-A and lung surfactant protein D (SP-D) are mainly found in the surfactant lining the pulmonary epithelial cells produced by alveolar type II cells and clara cells, but are also produced by epithelial cells lining the gastrointestinal tract [1]. Mannan-binding lectin (MBL), conglutinin and collectin-43 (CL-43) are serum proteins produced by the liver. So far conglutinin and CL-43 have only been found in Bovidae. Collectin liver 1 (CL-L1) is the latest characterised collectin found mainly in liver as a cytosolic protein [2]. The collectins are structurally related to C1q, a subcomponent of the complement component C1, and to ficolin [3]. Ficolin has a fibrinogen domain attached to a collagen-like region and exhibits sugar-binding activity [4], [5], [6] which is not found in C1q. When analysed by electron microscopy SP-A [7] as well as MBL [8], [9] and C1q appear as bouquet structures with arm lengths of 20 nm. SP-A oligomers mainly have six subunits, although smaller oligomeric forms have been described [10]. The degree of MBL oligomerisation varies from dimers to hexamers of subunits [8]. Like the rest of the collectins SP-A is involved in innate, non-adaptive immune defence. The collectins bind to microbial surface carbohydrates, inducing aggregation and thereby hinder infection or mediating phagocytosis through specific receptors on phagocytes.

Human SP-A is a polypeptide of 228 amino acid residues with a deduced mass of 24 kDa. SDS–PAGE analyses in the reduced state show a mass of 28–36 kDa [11]. The differences between the observed and deduced masses are mainly due to hydroxylation and N-glycosylation in the collagen-like region and the CRD, respectively [12]. Further heterogeneity comes from different alleles [13], [14] and the possibility of N-terminal extensions of one or three residues [15].

A duplication of the human SP-A gene appears to have occurred recently. Two human SP-A genes (SP-A1, SP-A2) corresponding to two different SP-A cDNA sequences have been analysed [12], [16], [17]. Moreover, an additional human SP-A pseudogene (SP-Aψ) has been characterised, which contains sequences highly homologous to exons encoding the α-helical neck region and the CRD of SP-A [18]. The nucleotide sequences of the two human SP-A genes differ only little and the resulting proteins deviate from each other at only 6–11 residues [12], [16], [17], [19], [20], most of which are located in the collagen-like region encoded by the first and second translated exon. The most significant difference is an extra cysteine residue in the SP-A1 gene product at position 85, between the interruption of the collagen-like region and the α-helical neck region. This cysteine residue may participate in interchain disulfide cross-linking of polypeptide chains of the same subunit [7]. Since both gene products are apparently necessary for the formation of correctly assembled SP-A in vitro, it has been suggested that the SP-A subunit is a heterotrimer composed of two SP-A1 gene products and a single product derived from the SP-A2 gene.

Alveolar proteinosis is a chronic disease of unknown pathogenesis. It is characterised by the production and accumulation of excessive surfactant in the alveoli and terminal bronchia of the lung [21]. Lung function of patients with alveolar proteinosis is maintained by lung lavage treatment, and SP-A isolated from lung lavage of these patients is known as proteinosis SP-A (protSP-A). Although significant differences between the molecular sizes of protSP-A and normal SP-A exist in solution, the only apparent difference between normal SP-A and protSP-A upon reduction, is that protSP-A exists as a monomer as well as a dimer [22], [23], [24]. Under reducing conditions, protSP-A has the same electrophoretic mobility, immunological determinants, and peptide map as SP-A isolated from normal human broncioalveolar lavage (BAL).

In this paper, we describe the structural characterisation of human SP-A, purified from BAL obtained from a patient suffering from pulmonary alveolar proteinosis.

Section snippets

Purification of human alveolar proteinosis SP-A

SP-A was purified from 3 l human bronchioalveolar lavage (BAL) obtained from a patient suffering from pulmonary alveolar proteinosis. Purification of SP-A was performed on a computer monitored FPLC system (FPLCdirector Version 1.3, Amersham Pharmacia Biotech). The 3 l of BAL without cells were centrifuged at 300×g and the pellet was dissolved in TBS containing 10 mM EDTA, 0.05% emulfogen. After incubation overnight at 4°C a 10 000×g centrifugation was performed. The supernatant was dialysed

Molecular mass of reduced proteinosis SP-A

Mass spectrometric analysis of the intact protSP-A did not yield any result. The protein was reduced and alkylated with iodoacetamide and purified by RP-HPLC. The resulting chromatogram showed a single peak with a tailing shoulder. The MALDI mass spectrum of the main chromatographic peak showed a wide peak with a mass distribution from 26.4 to 30 kDa (Fig. 1A). MALDI-MS analysis of the HPLC shoulder peak displayed the same mass peak with the addition of smaller peaks around 22.5–24.2 and 24.6

Discussion

In the present report, the primary structure of lung surfactant protein A isolated from a single proteinosis patient has been established. Like previously analysed collectins, SP-A is a very heterogeneous molecule [29], [30], [31], [32]. This is evident both when analysed by SDS–PAGE and upon mass spectrometry of the reduced intact molecule (Fig. 1). The heterogeneity is caused mainly by variations in post-translational modifications, but also by differences in the primary structure due to

Acknowledgements

We thank Ida Thornoe and Inger Christiansen for valuable assistance. This work was supported by the Novo Nordisk Foundation and the Benzon Foundation.

References (42)

  • K Ohtani et al.

    J. Biol. Chem.

    (1999)
  • J Lu et al.

    Immunobiology

    (1998)
  • Y Endo et al.

    Genomics

    (1996)
  • T Ohashi et al.

    J. Biol. Chem.

    (1997)
  • J Floros et al.

    J. Biol. Chem.

    (1986)
  • T Voss et al.

    Biochim. Biophys. Acta

    (1992)
  • G.F Ross et al.

    Biochim. Biophys. Acta

    (1987)
  • C.R Merril et al.

    Methods Enzymol.

    (1983)
  • T.N Krogh et al.

    Anal. Biochem.

    (1999)
  • S.N Bhattacharyya et al.

    Biochim. Biophys. Acta

    (1980)
  • Y.M Lee et al.

    J. Biol. Chem.

    (1991)
  • H Munakata et al.

    Biochim. Biophys. Res. Commun.

    (1982)
  • J.F van Iwaarden et al.

    J. Biol. Chem.

    (1992)
  • S Rubio et al.

    J. Biol. Chem.

    (1995)
  • J Lu et al.

    Biochem. J.

    (1996)
  • T Voss et al.

    Am. J. Respir. Cell Mol. Biol.

    (1991)
  • J.H Lu et al.

    J. Immunol.

    (1990)
  • S.B Laursen et al.

    Glycobiology

    (1995)
  • T.P Hickling et al.

    Mol. Med.

    (1998)
  • J.A Whitsett et al.

    Pediatr. Res.

    (1985)
  • R.T White et al.

    Nature

    (1985)
  • Cited by (23)

    • Chemical chaperone 4-phenylbutyric acid alleviates the aggregation of human familial pulmonary fibrosis-related mutant SP-A2 protein in part through effects on GRP78

      2018, Biochimica et Biophysica Acta - Molecular Basis of Disease
      Citation Excerpt :

      Usually, SP-A polypeptide chains can assemble into homo- or hetero-trimers and then six trimers can further build up to the octadecamers. SP-A also undergoes glycosylation [14], acetylation [15], and hydroxylation [16,17] modification. Although both genes are expressed primarily in lung alveolar type II cells, SP-A2 seems more biologically active than SP-A1 in most in vitro assays investigated [13].

    • Lectin-mediated binding and sialoglycans of porcine surfactant protein D synergistically neutralize influenza A virus

      2018, Journal of Biological Chemistry
      Citation Excerpt :

      Previously, profiling by fluorophore-assisted carbohydrate electrophoresis (FACE) analysis of the N-glycan present in the CRD of pSP-D derived from porcine BAL also indicated a complex N-glycan with strong heterogeneity in size (22). The only other detailed reports on glycan structures present on collectins describe the structure of the N-linked glycan that is conserved in the collagen domain of hSP-D (34) and the N-linked glycan present in the CRD of SP-A (35, 36), both complex-type glycans. The bi-antennary nonsialylated N-glycan in the collagen was shown not to be involved in interactions with IAV, and it most likely fulfills a role in assembly and stabilization of the collagen domain of SP-D.

    • Humanized SFTPA1 and SFTPA2 transgenic mice reveal functional divergence of SP-A1 and SP-A2: Formation of tubular myelin in vivo requires both gene products

      2010, Journal of Biological Chemistry
      Citation Excerpt :

      However and of interest, in a recent study, the ratio of SP-A1 to total SP-A in BAL samples from human subjects was found to vary depending on the age and/or health status of individuals (25). A predominance of SP-A2 products in BAL fluid has also been observed in a single patient with alveolar proteinosis (65). Furthermore, it is unknown how a varied SP-A1 and SP-A2 expression affects TM content and its potential significance in lung health.

    • Domains of surfactant protein A that affect protein oligomerization, lipid structure and surface tension

      2001, Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology
    View all citing articles on Scopus
    View full text